Character of Ocular Surface Mucins and Their Alteration in Dry Eye Disease

Character of Ocular Surface Mucins and Their Alteration in Dry Eye Disease ILENE K. GIPSON, PHD, YUICHI HORI, MD, AND PABLO ARGÜESO, PHD ABSTRACT At the ocular surface, three types of mucins are present. The large gel-forming mucin MUC5AC is expressed by conjunctival goblet cells. Some cells of the lacrimal gland acini express the small soluble mucin MUC7. The corneal and conjunctival epithelia express the membrane-associated mucins MUCs 1, 4, and 16. With the characterization of the mucin gene repertoire of the ocular surface epithelia, studies of the function of specific mucins, their gene regulation, and their alteration in ocular surface disease have begun. Current information suggests that all the mucins are hydrophilic and play a role in maintenance of water on the surface of the eye. The large secreted mucins represent the “janitorial service” that moves over the surface of the eye to wrap up and remove debris. The membrane-associated mucins form the glycocalyx, which provides a continuous barrier across the surface of the eye that prevents pathogen penetrance and has signaling capabilities that influence epithelial activity. Factors regulating mucin gene expression include retinoic acid, serum, and dexamethasone. Alteration in both secreted and membrane-associated mucins occur in drying ocular surface diseases. In Sjogren syndrome, MUC5AC expression is reduced, and in non-Sjogren dry eye, glycosylation of MUC16 appears to be altered. The pattern of expression of enzymes that glycosylate mucins is altered in ocular cicatricial pemphigoid. Therapies being evaluated for dry eye, including cyclosporine A, P2Y2 agonists, Accepted for publication February 2004. From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. Supported by: NIH/NEI Grants Nos. R01 EY03306 (IKG) and R01 EY014847 (PA). The authors have no proprietary interest in any product or concept discussed in this article. Single copy reprint requests to Ilene K. Gipson, PhD (address below). Corresponding author: Ilene K. Gipson, PhD, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114; Tel: 617-912-0210; Fax: 617912-0126; Email: [email protected]. Abbreviations are printed in boldface where they first appear with their definitions. ©2004 Ethis Communications, Inc. All rights reserved.

gefarnate, 15-(S)-HETE, and corticosteroids, may be efficacious due to their effect on mucin gene expression and secretion. KEYWORDS: dry eye, epithelia, epithelial mucins, mucins, MUC1, MUC4, MUC16, MUC5AC, ocular surface

I. INTRODUCTION he surface of the eye is covered by a tear film that serves to protect and lubricate the ocular surface, as well as to provide the major refractive surface for the visual system. The epithelial surface of the eye and its specialized glandular infoldings produce the components of the tear film that include water, protective antimicrobials, cytokines, lipids, and mucins (Figure 1A). In addition, the apical surface of the ocular surface epithelia, both corneal and conjunctival, provide a specialized interface between the tear fluid and the epithelium that stabilizes the fluid layer. That interface includes the undulating membrane ridges on the apical cell’s apical membrane, termed microplicae (Figure 1B), and emanating from their apices, a layer termed the glycocalyx (Figure 1C). Mucins are present in the glycocalyx layer, as well as in solution within the tear fluid. Mucins are defined as glycoproteins that are heavily glycosylated, with 50–80% of their mass comprised of carbohydrate. A second characteristic of mucins is the presence in their protein backbones of tandem repeats of amino acids that are rich in serine and threonine, which provide the sites for O-type glycosylation.1,2 The heavy glycosylation of mucins is believed to impart a highly negative charge and a hydrophilicity that provides a barrier to pathogen adherence and penetrance into the epithelium.3 It also provides a lubricating surface that prevents epithelial-epithelial adherence during the blink. Before molecular techniques were applied to the study of mucins, little was known of their biochemical character and their cellular sites of synthesis. These techniques applied to the study of mucins in the last decade have released a flood of new information that has helped greatly in our understanding of the ocular surface. This review will first summarize the current understanding of mucin protein structure, types, and function. It will then describe the distribution and pattern of expression of mucin genes by the ocular surface epithelia, and, lastly, dis-

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MUCINS AND THEIR ALTERATION IN DRY EYE / Gipson, et al OUTLINE I. Introduction II. Mucin protein structure A. Secreted mucins 1. Gel-forming mucins 2. Small soluble mucins B. Membrane-associated mucins III. Patterns of expression of mucins in ocular surface epithelia A. A major mucin of the conjunctival goblet cells: the gel-forming mucin MUC5AC B. Membrane-associated mucins of the stratified cells of corneal and conjunctival epithelia C. Lacrimal glands and accessory lacrimal glands as sources of mucin D. Relationship of ocular surface epithelial mucins to the tear film IV. Methods of assay of mucins in normal subjects and dry eye patients A. Impression cytology for mucin mRNA and protein analysis B. Tear collection for ELISA and immunoblot analysis of mucins C. Biopsy of conjunctiva for immunolocalization and in situ hybridization V. Alteration of mucins in dry eye A. Decreased levels of goblet-cell-associated mucin MUC5AC in dry eye B. Alterations of membrane-associated mucins in dry eye C. Glycosylation of mucins: Alterations in dry eye VI. Dry eye therapeutics and their effect on mucin production/distribution A. Cyclosporine A B. P2Y2 agonist C. 15-(S)-HETE D. Gefarnate E. Corticosteroid F. Autologous serum G. Vitamin A VII. Summary: Hypothesis regarding mucin alterations in dry eye

cuss the relationship of the mucins to the tear film. These summaries will introduce the reader to the work that has been done to characterize alterations of mucins in dry eye and also will allow development of new hypotheses for the role of mucins in health and drying ocular surface diseases. A comprehensive review on the topic of mucins of the ocular surface epithelia has recently been published.4 II. MUCIN PROTEIN STRUCTURE Before the molecular characterization of mucins, all mucins were considered to be secreted from specialized cells—usually goblet cells or mucus-secreting cells of 132

glands. Based on recent sequence data, however, it is now known that, in addition to secreted mucins, membraneassociated mucins are present at the apical surfaces of epithelia.1,2 Table 1 summarizes the mucins identified to date, along with several of their characteristics. Figure 2 demonstrates the major structural features of the secreted and membrane-associated mucins. To date, seven mucins are considered as secreted mucins, ten are membrane-associated, and several mucins remain uncharacterized. Human mucins are designated by “MUC” followed by a number that indicates the chronological order of their discovery.

Figure 1. A. Diagram of the ocular surface and adnexia, showing (in various colors) epithelial sites of mucin expression and the tear film. B and C. Scanning electron micrograph B and transmission electron micrograph C of surface ridges or microplicae on the corneal (B) and conjunctival surface (C). Note electron-dense glycocalyx (arrow) at tips of microplicae in C, as well as actin filaments inserting into the same tips from the cytoplasmic face. Bar in B = 10 μm; bar in C = 0.1 μm. (A is reprinted from Gipson and Argüeso4 with permission of Int Rev Cytol. C is reprinted from Nichols et al52 with permission of the authors and Invest Ophthalmol Vis Sci.)

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For example, MUC1 was the first of the human mucin genes to be cloned and characterized. Mouse homologues to human mucin genes are designated by “Muc” and rat genes by “rMuc,” while the nascent unglycosylated mucin protein is termed “apomucin.” A. Secreted Mucins

Two types of secreted mucins have been identified (Figure 2A)—the large gel-forming mucins and the smaller soluble mucins. (For review, see Gendler and Spicer, 1995,1 and Moniaux et al, 2001.2) 1. Gel-forming Mucins Five large gel-forming mucins have been described; four are encoded on chromosome 11p15.5, and a recently discovered one is present on chromosome 12q12. The gel-forming mucins encoded on chromosome 11p15.5 include MUCs 2, 5AC, 5B and 6, and the one on chromosome 12q12 is MUC19. These mucins are called gel-forming because they are responsible for the rheological properties of mucus.5 They share common structural motifs and are believed to have evolved from a common ancestral gene, similar to the human von Willebrand factor gene—a component in blood that facilitates clotting.6,7 These gel-forming mucins are expressed by goblet cells of the respiratory, gastrointestinal, endocervical, and ocular surface epithelia, as well as by the mucin cells of the submucosal glands associated with these epithelia. The goblet cells or mucus-producing cells of each of these organ systems have a tissue- and cell-specific pattern of expression of the gel-forming mucins (For review, see Gipson and Argüeso, 2004.4) At the ocular surface, the major mucin of this class expressed by the goblet cells of the conjunctiva is MUC5AC (see below).8 The gel-forming mucins are probably the largest glycoproteins known. This is borne out by the size of their cDNAs, which range from 15.7 to 17 kb, making the molecular weights of these huge apomucins approximately 600 kDa.2 The gel formers share several structural motifs (as an example, see diagram of MUC5AC, Figure 2). Each has a large, central, tandem repeat domain flanked by cysteine-rich domains that have homology to the so-called D domains of von Willebrand factor. Three cysteine-rich D domains are present on the amino terminal side of the central tandem repeat, and one on the carboxy terminal side (except for MUC6, which lacks the C-terminal D domain). These domains allow multimerization of individual mucin molecules through disulfide bond formation, which begins in the endoplasmic reticulum, with further multimerization in the Golgi apparatus.9,10 Multimerization and subsequent glycosylation produces huge macromolecular structures with an estimated molecular weight of up to 40 MDa.10 2. Small Soluble Mucins Mucins in the second category of secreted mucins are the small soluble mucins that include MUC7 (Figure 2A) and MUC9 (also known as oviductin, whose only demon-

Figure 2. Diagrams demonstrating structural features of the mucins expressed by ocular surface epithelia. All mucins have tandem repeats (TR) of amino acids in their protein backbone that are rich in serine and threonine, which are O-glycosylated (YYY). Repeats of TRs vary in individuals and alleles. A: Of the secreted-type mucins, MUC5AC, expressed by goblet cells, is a large gel-forming mucin, with D domains that are rich in cysteines and that provide disulfide cross-linking sites for polymerization of these mucins to form the mucous network that gives mucus its rheologic properties. The small soluble mucin MUC7, expressed by acinar cells of the lacrimal gland, has a histatin-like domain (Hsn) in the amino terminal region and does not form disulfide linkages. B: The three membrane-associated mucins, MUCs 1, 4 and 16, are expressed by corneal and conjunctival epithelia. MUC1’s cytoplasmic tail has seven tyrosine residues that can be phosphorylated (P) and participate in signal transduction, as well as sites of association with β and γ catenin, which link to the cytoskeleton. MUC4 has EGF-like domains extracellularly. All membrane-associated mucins have cleavage sites (*) associated with shedding of the ectodomain from the surface of the cell.

strated tissue expression is fallopian tube).11 These mucins lack cysteine-rich D domains and are present predominantly as monomeric species. MUC7 was originally cloned from salivary gland and is one of the smallest mucins known, comprised primarily of tandem repeats of amino acids. It has a histatin-like domain on the N-terminal side of the tandem repeat and lacks cysteine-rich domains. The MUC7 apomucin is 39 kDa,12 which is secreted by serous cells rather than mucus cells of both the salivary glands13

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and the submucosal glands of the bronchial airways,14 as well as by acinar cells of the lacrimal gland.15 B. Membrane-associated Mucins

As stated above, until molecular techniques were applied to the characterization of mucins, all mucins were assumed to be of the secreted type; however, increasing numbers of mucins are being characterized as having hydrophobic membrane-spanning domains near their carboxy terminus (Figure 2B). These mucins appear to be major constituents of the glycocalyx of cells of all wet-surfaced epithelia—including both simple and stratified—and are estimated to extend 200– Table 1.

500 nm from the cell surface.16 MUCs 1, 3A, 3B, 4, 12, 13, 15 16, 17, and 20 have been designated as membrane-associated (Table 1). Genes of four of this group (MUCs 3A, 3B, 12, and 17) are clustered on chromosome 7q22 (Table 1).1719 Of the membrane-associated mucins, MUCs 1 and 4 have been the most extensively studied (for reviews, see Gendler, 2001,20 and Carraway et al, 200021). The major part of the extracellular portion of the molecule is occupied by the heavily glycosylated tandem repeat domain that in some instances extends nearly to the amino terminus (Figure 2B). These extracellular domains, also called ectodomains, are envisioned as extending from the cell surface to form the glycocalyx.

Human Epithelial Mucin Genes

(Those whose expression in ocular surface epithelia has been verified by both mRNA and protein assays, and whose cellular origin is known are shown in boldface.)

Amino Acids in Tandem Repeat References

Type if Sequence Verified

cDNA Clone Source

Chromosomal Mapping

MUC1*

Membrane-associated

Mammary/pancreatic tumor

1q21-q23

20

112,113

MUC2

Gel-forming/secretory

Intestine

11p15

23

114

MUC3A*Δ

Membrane-associated

Intestine

7q22

17

115

MUC3B*Δ

Membrane-associated

Intestine

7q22

17

18

MUC4Δ

Membrane-associated

Trachea

3q29

16

116

MUC5AC

Gel-forming/secretory

Trachea

11p15

8

117,118

MUC5B

Gel-forming/secretory

Trachea

11p15

29

119

MUC6

Gel-forming/secretory

Stomach

11p15

169

120

MUC7

Soluble monomer/secretory

Salivary gland

4q13-q21

23

12

Trachea

12q24.3

13/41

121

Fallopian tube

1p13

15

122

Intestine

7q22

28

17

Designation

MUC8 MUC9

Secreted, 120 kDa

MUC11 MUC12*Δ

Membrane-associated

Intestine

7q22

28

17

MUC13*Δ

Membrane-associated

Intestine, trachea

3q13.3

15

28

MUC15

Membrane-associated

Mammary gland

11p14.3

None

123

MUC16*

Membrane-associated

OVCAR-3 cells

19p13.2

156

39

MUC17*Δ

Membrane-associated

Intestine

7q22

59

19

MUC19

Secreted

Genomic databases

12q12

MUC20

Membrane-associated

Kidney

3q29

124 19

30

Characteristics of Mucins: * Have SEA Domains (MUC16 has six SEA Domains—five of them in the tandem repeat region.) Δ Have EGF-like Domains (MUCs 3A, 3B, & 4 have two EGF-like domains.) Note: MUCs 14 and 18 do not appear in the HUGO gene nomenclature website: www.gene.ucl.ac.uk/nomenclature, then Quick Gene Search MUC.

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Experimental studies indicate that the extracellular, highly glycosylated tandem repeat domain functions as a “disadhesive,” preventing cell-cell and cell-matrix interactions.22 The membrane-associFigure 3. In situ hybridization demonstrating message for MUC5AC in human conjunctival goblet cells ated mucins do not have (A) and immunohistochemical localization of MUC5AC protein to human conjunctival goblet cells (B). cysteine-rich domains and Inset in B shows staining of individual mucin packets. Bars = 50 μm. (Reprinted from Gipson and do not form multimers. Argüeso4 with permission of Int Rev Cytol.) They have short cytoplasmic domains (Figure 2B), and agrin (SEA) module at a region that has the sequence which in MUC1 is conserved between mammalian speG/SVVV for MUC135,36 and rMuc3.37 Several of the other cies and is reported to be associated with the actin cyto23,24 There is also growing evidence that the cytomembrane-associated mucins that have SEA domains inskeleton. plasmic domains interact with cytoplasmic proteins to faclude MUCs 3A, 3B, 12, 13, 17,16-18,28,38 and 16,39 which has six SEA domains. After reassociation of the α and β cilitate signal transduction. Tyrosine residues in the cytosubunits, the heterodimer moves through the Golgi for plasmic domains of MUC1 can be phosphorylated, and, glycosylation and then to the apical membrane of epithethus, through interaction with proteins having SH2 dolial cells. It is not clear whether the shedding of the mains, are believed to participate in signal transduction ectodomain of the membrane-associated mucins occurs that mediates cell cycle and apoptotic activity.25,26 Putative tyrosine phosphorylation sites are also present in MUCs at the association site of the α and β subunits or whether 12 and 16.17,27 shedding involves a different proteolytic event. The In addition to their function as “disadhesives” and sigectodomain of MUCs 1, 4, and 16 have been detected in nal transducers, the membrane-associated mucins may tear fluid (Spurr-Michaud et al and Hori et al, submitted have other cytokine-like functions. MUCs 3A, 3B, 4, 12, to ARVO, 2004). 13, and 17 have two or more epidermal growth factor III. PATTERNS OF EXPRESSION OF MUCINS IN (EGF)-like domains between the tandem repeat region and OCULAR SURFACE EPITHELIA the membrane-associated domain.17-19,21,28 Studies of The epithelium that covers the entire ocular surface rMuc4 have provided evidence that the EGF-like domains expresses mucins that contribute to maintenance of the play a role in the regulation of epithelial growth (for retear film. Not only do the goblet cells of the conjunctiva view, see Carraway et al, 200229). The EGF domain of rMuc4 interacts with ErbB2 growth factor, inducing ErbB2 produce mucins, the stratified epithelia of both cornea and phosphorylation and potentiating neuroregulin-activation conjunctiva, as well lacrimal gland acinar and ductal cells, of ErbB3 receptor. These receptor interactions suggest that produce mucins. The pattern of expression and type of rMuc4 has the potential to be involved in regulation of mucin produced in the various regions of the ocular surepithelial cell growth. face do, however, vary and may reflect the functions of Several of the membrane-associated mucins, MUCs 1, each epithelial zone. 4, 16, and 20, are present in both a membrane-associated A. A Major Mucin of the Conjunctival Goblet Cells is and a soluble form.29-31 The soluble form may be the rethe Gel-forming Mucin MUC5AC sult of splice variants in which the membrane-associated Since goblet cells of the conjunctival epithelium have domain is post-transcriptionally removed2,20,30 or the ectodomain or α domain of the mucins is shed from the long been considered the major source of the mucus on surface of cells.32 MUC1, MUC4 and MUC16 all appear the ocular surface, it is not surprising that they appear to to be shed from the epithelial surface.31,33,34 be the major source of the gel-forming mucins of the tear The mechanism by which the membrane-associated film. As described above, the gel-forming mucins are the mucins are shed from the apical surface of cells is unclear. largest of the mucins and give mucus its rheological propApomucins of MUC1, rMucs 3 and 4 are proteolytically erties. It seems reasonable that the large gel-formers for cleaved in the endoplasmic reticulum where they reassothe tear film be made in and secreted from the conjuncticiate to form noncovalently bound heterodimers composed val goblet cells, since they can secrete directly onto the of an α (extracellular tandem repeat domain) and a β (inocular surface without having to send their large secreted cludes EGF-like domains, transmembrane domain, and product through a duct system. As demonstrated by in cytoplasmic tail) subunit. The proteolytic cleavage site that situ hybridization (ISH) and immunofluorescence microsproduces the subunits is hypothesized to be at a GDPH copy, the goblet cells of the human conjunctival epithesequence for rMuc4 (for review, see Carraway et al, 200021), lium express the gel-forming mucin MUC5AC (Figure or the so-called sea urchin sperm protein, enterokinase 3).8,40 Fluorescence in situ hybridization shows that the THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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message for the mucin is located near the goblet cell nucleus, corresponding to the position of the major accumulation of the endoplasmic reticulum of the cell. All the goblet cells in sections of human bulbar conjunctiva appear to bind probes to the MUC5AC gene (Figure 3A), but there has not been a systematic study of all zones of the human conjunctiva, nor have there been studies of the goblet cell crypts located in the forniceal region of the human conjunctiva.41 Antibodies specific to the D domain of MUC5AC have been developed and their specificity demonstrated.42 These antibodies localize to the secretory mucin packets of the conjunctival goblet cells (Figure 3B), and they have been used in an ELISA to detect and measure levels of MUC5AC in tears. Pretreatment of tear proteins with neuraminidase to remove terminal sugars enhances the binding of the antibody. Although there is considerable variation from one individual to another in amount of MUC5AC in tears, the levels of the mucin are consistent within an individual over several samplings.42 Screening for the expression of three other gel-forming mucins, MUCs 2, 5B, and 6, in conjunctival epithelium has been done by Northern blot and polymerase chain reaction (PCR), but to date there have been no assays for MUC20. Low levels of MUC2 message have been detected by PCR (some 5,600-fold lower than 5AC) in conjunctival epithelial RNA of some individuals,40,43 but, despite attempts to demonstrate the localization of the MUC2 message by in situ hybridization in both human and rat conjunctiva, no message has been detected. Thus, it is not known if the MUC2 message is translated.8 PCR has also detected MUC5B message in the conjunctiva, but neither the message nor protein has been demonstrated in the goblet cells.15 B. Membrane-associated Mucins of the Stratified Cells of Corneal and Conjunctival Epithelia

The human ocular surface epithelia express at least three membrane-associated mucins—MUCs 1, 4 and 16 (Figure 4).44-46 Presence of these three mucins has been verified at both the mRNA and protein levels, using Northern blot analysis or PCR, in situ hybridization, immunohistochemistry, and immunoblot analysis. Multiple methods are necessary for verification of expression of mucins, first, because low levels of mucin mRNA may not be translated, and because nonspecific binding of proteins (e.g., antibodies) to mucins is well known. The measure of relative RNA levels of the three membrane-associated mucins in conjunctival samples by real-time PCR suggest that MUC1 and MUC16 transcripts are the lowest in number, as compared to MUC4 mRNA levels.4 Difficulty in obtaining human corneal epithelial samples has prevented similar comparisons for corneal epithelium. In situ hybridization has demonstrated that message for MUC1 is dispersed throughout the corneal and conjunctival epithelia, but immunolocalization studies demonstrate that the protein is present only in the apical cell membranes.45 In conjunctival epithelium, the mucin ap136

pears to be present on apical as well as subapical cells. In both epithelia, detection of the mucin with monoclonal antibodies specific to MUC1 is enhanced by pretreatment of tissue sections with neuraminidase to remove terminal sugars on the mucin.45 Unlike MUC1, there is regional variation of expression of mRNA for MUC4 across the ocular surface epithelia. In sections in which conjunctiva, limbus, and peripheral cornea are all present (Figure 4), message levels appear the greatest in the conjunctival and limbal epithelia, with a noted decrease in peripheral cornea and a greater diminution toward central cornea. Assay of levels of MUC4 message in central corneal epithelium by Northern blot also demonstrates lower message levels of MUC4 in central compared to peripheral corneal epithelium.47 Like the message distribution for MUC1, MUC4 mRNA appears in all cell layers in conjunctival and peripheral cornea, but there appears to be a greater intensity of S35labeled MUC4 probe in apical cells.48 Immunolocalization of MUC4 in the conjunctival and corneal epithelia does not show the discrete membrane pattern of localization that other membrane-associated mucins do. Antibody to ASGP1 (HA-1), which recognizes the carboxy terminal region of the α subunit of rMuc4, binds to the cytoplasm of the entire stratified epithelium (see Figure 8 in Pflugfelder et al, 200047). Recent data indicate that the stratified cells of the corneal and conjunctival epithelia express the recently cloned, membrane-associated mucin MUC16.46 This mucin was cloned and sequenced as the result of a long, 20-year effort to characterize the ovarian tumor marker CA125.27,39 In situ hybridization studies demonstrate MUC16 message is in apical cells, as well as some suprabasal cells in cornea and conjunctival epithelium (Figure 4). Immunolocalization studies using antibodies specific to the mucin demonstrate that the glycoprotein is localized along the apical surface of both cornea and conjunctiva, in the position of the glycocalyx (Figure 4). MUC16 carries a carbohydrate epitope recognized by a monoclonal antibody designated H185 (see Argüeso et al, 200346), and by immunoelectron microscopy, H185 localizes to the tips of the microplicae, in the position of the glycocalyx (Figure 5). The distribution of this antibody is altered on the ocular surface of patients with non-Sjogren dry eye (see below and Danjo et al, 199849). Assay for other membrane-associated mucins by PCR methods has demonstrated low levels of MUC11 mRNA (Gipson and Argüeso, unpublished data), but neither immunolocalization nor in situ hybridization studies have verified the presence of the mucin to date. PCR assays for MUCs 3, 12, 13 and 17 in corneal and conjunctival epithelia to date have been negative (Gipson et al, unpublished data). Evidence exists that the extracellular domains of each of the membrane-associated mucins MUCs 1, 4, and 16 are shed into the tear film. Pflugfelder et al report detection of both ASGP1(α subunit) and ASGP2 (β subunit) in tears.47 In several preliminary studies, all three of the

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mucins have been detected in tears (Spurr-Michaud et al and Hori et al, submitted to ARVO, 2004). C. Lacrimal Glands and Accessory Lacrimal Glands as Sources of Mucin

Recent studies by Jumblatt et al15 have demonstrated MUC7 mRNA and protein in four of six specimens of human lacrimal tissue RNA. Their in situ hybridization studies have demonstrated presence of message in some of the acinar cells of the gland. Protein for the small soluble mucin has also been detected in cellular extracts of the tissue by immunoblot, but curiously, the mucin has not been detected in tear samples assayed by immunoblot. 15 PCR analysis performed in the same study has identified message for MUCs 1 and 4, and also identified message for MUC5B in one sample, but in situ hybridization studies localizing the message of these mucins have not been done. rMuc4 has also been reported to be produced by rat lacrimal gland in both membrane and soluble forms.50 To date, the muFigure 4. A and B demonstrate message and protein localization, respectively, of MUC1 in cins of the lacrimal gland appear to be human corneal epithelium. C shows that mRNA localization of MUC4 diminishes from coneither the small soluble mucins or junctiva on the left, across the limbus (single arrow) toward the cornea on the right (two arrows). D and E show fluorescence in situ hybridization of MUC16 mRNA in cornea and membrane-associated mucins. conjunctiva, respectively, and F and G show immunohistochemical localization of MUC16 Accessory lacrimal glands, the protein in human cornea and conjunctiva, respectively. Bars in A,C = 100 μm; Bars in B,Dglands of Klaus and Wolfring, have G = 10 μm. (Reprinted from Gipson and Argüeso4 with permission of Int Rev Cytol.) cellular characteristics consistent with mucin secretion. Histologically, they mucins expressed by the apical cells. Upon high magnifiappear to be a mixed population of cells, with both serous cation, striations within the glycocalyx can be seen (Figand mucus types of secretory vesicles—an appearance ure 1C), and each probably represents individual, highly much like that of other submucosal glands.51 These glands are, in all probability, producing mucins but, to date, they glycosylated, membrane-associated mucins, which are eshave not been examined for mucin production. timated to extend 200–500 nm from the cell membrane.16 Nichols’ high resolution electron microscopy also shows D. Relationship of Ocular Surface Epithelial Mucins that actin cytoskeletal filaments insert into the tips of the to the Tear Film microplicae and extend into the cytoplasm (Figure 1C).52 Of all the wet-surfaced epithelia of the body, the ocuPerhaps these actin filaments are the site of interaction lar surface is the most accessible, and, thus, this fluidwith the cytoplasmic domain of the membrane-associated epithelial interface can be readily observed and studied. mucins.23 Localization of the monoclonal antibody H185, which recognizes a carbohydrate epitope carried by the In spite of accessibility, the character, thickness, and dismembrane-associated mucin MUC16, by immunoelectron tribution of mucins in the fluid are debated. The tear fluidmicroscopy,46 shows labeling on the microplicae of hucell membrane interface over the apical cells of the guinea man corneal and conjunctival epithelia, both in vivo and pig conjunctiva, as captured by rapid freeze and electron in vitro (Figure 5A and B).53 These data verify the presmicroscopy, is demonstrated in Figure 1C. The apical ence of the membrane-associated mucin in the glycocalyx. membrane folds or the microplicae of the apical cells of The granular material above the glycocalyx in Figure both cornea and conjunctiva have at their tips an elec1C is assumed to be the gel-forming mucin secreted by tron-dense zone that interfaces with the tear film compothe conjunctival goblet cells, which, in its hydrophilic exnents.52 This electron-dense zone, or glycocalyx, has in it the extracellular domains of the membrane-associated tended network, houses other bactericidal proteins and THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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fluids secreted by the lacrimal and accessory lacrimal glands. A diagram depicting this arrangement is shown in Figure 5C. Early diagrams depict a three-layered tear film with a sharp mucus-aqueous layer interface, but, since mucins are highly hydrophilic and some are small and soluble, it is more likely that mucins mix throughout the aqueous phase of the tear film (Figure 5C). Studies using interferometry, as well as confocal microscopy, have suggested that the mucus component is thicker than once envisioned, with measurements ranging from 3–30 μm.54,55 Perhaps these methods include the microplicae with their associated glycocalyx layer composed of membrane-associated mucins that may extend 1–3 μm into the tear film. Also, since the mucus secreted from the goblet cell moves around over the surface of the eye and is cleared by blinking, that component of the tear film may vary, depending on the area of the palpebral fissure. Secreted mucus may be envisioned as the cleanup or “janitorial service” of the ocular surface; the fully hydrated mucin multimers move over the glycocalyx, wrapping up dirt, debris, and unwanted pathogens, then moves them off the eye through the nasolacrimal duct. In order to function as the cleanup, particle-moving blanket that mucus layers of all wet-surfaced epithelia provide, the layer must move freely over the surface. The eyelids, Figure 5. Immunoelectron microscopic localization of MUC16 on microplicae of during the blink, facilitate this process, movcultured human corneal epithelial cells (A) and native corneal epithelial cells ing the mucus layer and tear film toward the (B). Data shown in A and B support the diagram (C) of tear fluid composition and its association with membrane-associated mucins at the tips of the exiting nasolacrimal duct. The movement of microplicae. The diagram shows MUC5AC multimers in solution in the tear the mucus layer over the glycocalyx implies fluid, along with other protective molecules secreted by the lacrimal gland. that the mucins of the membrane-associated Negative charges on the mucins prevent their direct association and allow movement of goblet-cell-derived MUC5AC over the surface of the eye for its type are disadhesive, preventing strong interjanitorial duties of collecting and removing debris and harmful pathogens. (Reactions with the secreted mucins. Indeed, as printed from Gipson and Argüeso4 with permission of Int Rev Cytol.) stated earlier, experimental data indicate that the glycosylated tandem repeats of the membrane-associated mucin-actin cytoskeleton association. brane-associated mucins have disadhesive properties.22 Cells that first appear at the surface of the stratified IV. METHODS OF ASSAY OF MUCINS IN corneal epithelium during normal cell differentiation and NORMAL SUBJECTS AND DRY EYE PATIENTS turnover have numerous microplicae and, over time, as In the past, the major limitations to the assay of huthe cell ages, these membrane projections disappear.56 Antibodies to carbohydrate epitopes on membrane-assoman ocular mucins from tears or tissues have been the ciated mucins bind avidly to the newly emerged cells and small amount retrievable from the ocular surface and the their microplicae, and as the number of microplicae delack of appropriate biochemical tools to specifically idencreases on cells, so does the amount of antibody bindtify individual mucins. Several analytical techniques (i.e., ing.57 These data suggest that shedding of mucins occurs real time PCR, immunoassay) with high sensitivity and over time from the cell surface, leaving the oldest cells with a high specificity are now available for mucin analywithout both microplicae and membrane-associated musis. These techniques have been successfully used to idencins. The oldest cells may lose their disadhesive character tify, localize, and quantify individual mucin mRNAs and and, thus, adhere to the mucus of the tear film to be reproteins in samples collected from normal subjects and moved and disposed of by mucus entrapment and removal from patients with dry eye. This section summarizes rethrough the nasolacrimal duct. These data also suggest that cent research in the analysis of mucins collected by differthe microplicae structure is held in place by the mement methods from the ocular surface. 138

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MUCINS AND THEIR ALTERATION IN DRY EYE / Gipson, et al A. Impression Cytology for Mucin Mrna and Protein Analysis

Impression cytology is a simple, inexpensive, and noninvasive technique to collect epithelial cells from the ocular surface.58 After instillation of a topical anesthetic into the surface of the eye, a sterile impression disc (e.g., nitrocellulose, polyethersulfone) is applied on the bulbar conjunctiva for a short period of time and then carefully removed.59,60 This procedure allows the collection of apical and subapical cells of the conjunctival epithelium, as well as goblet cells. The method is particularly useful for the analysis of mucins, as membrane-associated and gelforming mucins are present in the suprabasal cells of the conjunctival epithelium and in the goblet cells, respectively. Conjunctival epithelial cells collected from a single individual can then be used to analyze mucin mRNA expression and mucin protein. Recent progress in cloning and characterization of mucin genes has facilitated the use of PCR to determine the mucin mRNA repertoire in cells collected by impression cytology. Because of the small amount of starting material, it is necessary to pool the nitrocellulose discs collected from both eyes of the subject. RNA isolation from the discs, reverse-trancription into cDNA, and real timePCR analysis of the mucin mRNA content can be determined as described previously.42 Using this method, the membrane-associated mucins MUCs 1, 4, and 16, as well as the gel-forming mucin MUC5AC, were found in a population of 18 normal individuals.42 The relative levels of MUC1 and MUC16 transcripts were the lowest in number, as compared to MUC4 mRNA levels.4 In addition, MUC7 mRNA and low levels of MUC2 mRNA have also been found using conventional PCR in conjunctival cells collected by impression cytology61; however, it is not known whether these transcripts are translated. Samples collected by impression cytology are also suitable for mucin protein analysis by immunofluorescence47,59,62 and flow cytometry.60 Immunofluorescence microscopy has been used to detect MUC4 in impression cytology discs collected from human cornea and conjunctival epithelia,47 as well as the H185 carbohydrate epitope on MUC16 from conjunctival epithelium.49 MUC5AC has been detected by flow cytometry in goblet cells from samples of conjunctival epithelium collected by impression cytology.60 B. Tear Collection for Elisa and Immunoblot Analysis of Mucins

The gel-forming mucin MUC5AC has been detected in tears from normal individuals, collected by micropipette or after extraction from Schirmer strips.42,63 In these studies, the relative amount of MUC5AC mucin was evaluated by ELISA or by direct immunoassay on the Schirmer strip, using polyclonal antibodies raised against synthetic peptides corresponding to nonglycosylated domains flanking the central tandem repeats in the MUC5AC molecule. The results showed that, when the tear samples were not

pretreated with glycosidases, the amount of MUC5AC was highly variable between individuals. Interestingly, despite the fact that the MUC5AC antibodies were against nonglycosylated regions, treatment with neuraminidase— a glycosidase that removes terminal sialic acid on glycoproteins—increased the binding of the antibody to the mucin, decreasing the variability between individuals.42 This result suggests that differential mucin glycosylation in some individuals masks recognition of the protein epitope recognized by the MUC5AC antibody. In semiquantitative studies, it is then useful to partially deglycosylate the sample to facilitate access of the antibody to the mucin apoprotein. The amount of membrane-associated mucin shed into the tear film by the ocular surface epithelia can be assessed by immunoblot. Pflugfelder et al have detected the two MUC4 subunits (ASGP1/α subunit and ASGP2/β subunit) in tears collected with porous polyester rods from normal volunteers, using immunoprecipitation techniques followed by SDS-PAGE and immunoblot analysis.47 In addition to MUC4, the presence of MUC1 in tears has also been detected by immunoblot analysis of Schirmer strips of human tear fluid.64 Surprisingly, despite the fact that both RNA and protein corresponding to the secreted MUC7 mucin are present in the normal human lacrimal gland and conjunctival tissue, MUC7 protein has not been detected in tears by immunoblot.15 Perhaps the concentration of MUC7 in lacrimal tears is too low to be detected with the colorimetric immunoblot technique. C. Biopsy of Conjunctiva for Immunolocalization and In Situ Hybridization

Collection of conjunctival biopsies from living donors for mucin analysis is a much more invasive method than impression cytology; however, it allows localization of the specific sites of mucin mRNA synthesis and protein expression through all the cell layers of the epithelium. Biopsies are obtained from the bulbar conjunctiva of patients who are undergoing cataract surgery. Distribution of mucin mRNA and protein is then analyzed by ISH and immunological techniques, respectively. It is always advisable to use a combination of ISH and immunohistochemistry to demonstrate mucin distribution, since detection of mucins exclusively by immunohistochemistry may be subject to error due to poor characterization of the mucin antibodies and to the “sticky” nature of mucins, which induces nonspecific binding.65 For ISH, the biopsy is normally fixed in 4% paraformaldehyde and embedded in paraffin.48 The presence of tandemly repeated sequences in the nucleotide sequence of mucins has facilitated their analysis by ISH, since probes to the tandem repeat bind at multiple sites along the mucin mRNA, enhancing the signal and facilitating mucin mRNA detection.48 The method is not quantitative, since mucin genes exhibit polymorphisms in the tandem repeat sequence.48 The presence and distribution of MUC1 and 16 transcripts have been demonstrated by ISH in corneal

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and conjunctival epithelia.45,46 MUC4 mRNA has been demonstrated in the stratified conjunctival and limbal epithelial cells but not central cornea, whereas MUC5AC trancripts are present in conjunctival goblet cells.8 MUC7 mRNA has been detected by ISH exclusively in the lacrimal gland.15 For immunohistochemistry, the biopsy can be frozen or embedded in paraffin, depending on the antibody. Antigen retrieval techniques, such as microwave, proteolytic digestion, or deglycosylation may be required to unmask the epitope recognized by the antibody.66 V. ALTERATION OF MUCINS IN DRY EYE Only with the recent development of methodologies has it become possible to measure specific mucins in ocular surface epithelia and tears of patients with drying ocular surface disease. The few studies that have been done indicate alteration in secreted mucin mRNA and protein, as well as alteration in distribution and/or Figure 6. Levels of MUC5AC mRNA (A) and protein (B) were significantly less glycosylation of membrane-associated mucins. in Sjogren patients (N = 11) as compared to normal subjects (N = 15-30) (Reprinted from Argüeso et al42 with permission of Invest Ophthalmol Vis Sci.) It has long been known that a decrease in the density of mucin-producing goblet cells in the conjunctival epithelium is a common characrats deprived of vitamin A for 20 weeks decreases, as did teristic in dry eye patients.62,67,69 As the disease progresses identifiable goblet cells. The possibility that another sein severity, the number of goblet cells decreases further, creted mucin increases remains a hypothesis to be tested. and squamous metaplasia and keratinization of the ocular These studies provide definitive evidence that the levsurface ensues. This led to speculation that mucus defiels of mRNA and protein of the gel-forming mucin ciency could be the direct cause of tear film instability.70,71 Initial studies measuring hexosamine and O-linked oliMUC5AC decreases in dry eye, correlating with a reducgosaccharides as indicators of mucin found a correlation tion in goblet cell density. Measurement of MUC5AC mubetween the content of mucin-like glycoprotein in tears cin in tears is a valuable noninvasive method to detect dry and goblet cell density in conjunctiva.70,71 Surprisingly, eye disorders and could potentially be used to evaluate the data also showed a substantial amount of mucin-like the effect of pharmaceutical products in the treatment of glycoprotein present, even when only few goblet cells were dry eye. present.70,71 Perhaps this mucin-like glycoprotein is shed B. Alterations of Membrane-associated Mucins in membrane-associated mucin, now measurable in tear fluid. Dry Eye A. Decreased Levels of Goblet-cell-associated Mucin In addition to the recent studies demonstrating alterMuc5ac in Dry Eye ation in the amount of the secreted mucin MUC5AC in Recent data using real-time RT-PCR have shown that Sjogren dry eye,42 membrane-associated mucins are althe number of RNA transcripts for MUC5AC in the contered in non-Sjogren dry eye.49 Danjo et al demonstrated a significant difference in the binding pattern of an antijunctival epithelium of patients with Sjogren syndrome body against a carbonate epitope antibody (H185) carried was significantly lower than in normal individuals.42 As measured by ELISA, the protein levels of MUC5AC were by MUC16 to conjunctival epithelium obtained by imalso significantly reduced in the tear fluid of the same papression cytology in normal eyes compared with those of tients, corroborating mRNA data obtained using real-time patients with non-Sjogren dry eye.49 In normal eyes, the antibody bound to apical cells in a mosaic pattern that RT-PCR (Figure 6). Additional evidence correlating a reshows various degrees of H185 binding to cells, as deterduction in MUC5AC mucin with dry eye is the reduction mined by immunofluorescence microscopy on impression in the percentage of MUC5AC-positive conjunctival cells cytology samples (Figure 7). The mosaic pattern was abin dry eye patients, as determined by flow cytometry.60 Tei et al have correlated a reduction in MUC5AC musent in patients with non-Sjogren dry eye. These data were cin gene expression with the goblet cell density, using an corroborated by immunoelectron microscopy (Figure 7B animal model of ocular surface keratinization, using vitaand D). The question arises—is the alteration in distribumin A-deficient rats.72 In this study, the number of tion of H185 antibody binding a result of lack of expresrMuc5AC transcripts in the ocular surface epithelium of sion of the membrane-associated mucins or in their glyco140

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sylation? We previously reported that the levels of MUC1 mRNA from conjunctival impression cytology showed a trend toward decreasing amounts in patients with Sjogren syndrome compared with normal subjects, but there was no statistically significant difference.42 Additionally, there was no difference in levels of MUC4 mRNA. A thorough study of the level of all the membrane-associated mucin protein in tears has not been done. Pflugfelder et al, using an antibody recognizing an epithelial membrane-associated mucin expressed in human conjunctiva (AMEM2), found less binding to the conjunctiva in patients with Sjogren syndrome than in normal subjects, as determined by immunohistochemistry analysis.62 Two pieces of data suggest that the alteration in H185 mucin distribution in dry eye is related to glycosylation in the membrane-associated mucins. Firstly, there is a loss of binding of the H185 antibody to apical cells of conjunctival epithelium in non-Sjogren dry eye. Secondly, in more advanced severe dry eye, such as in ocular cicatricial pemphigoid (OCP), there is alteration in distribution of glycosyl transferases that glycosylate mucins in the conjunctival epithelium, with complete loss of their expression in keratinized regions. Further investigation is needed to determine the level of membrane-associated mucins in tears of dry eye patients and factors affecting their expression.

Figure 7. Distribution of the H185 carbohydrate epitope carried by MUC16 is altered in non-Sjogren dry eye. The pattern of distribution in normal subjects is shown by immunofluorescence microscopy (A) and immunoelectron microscopy (B) of samples taken by impression cytology. Note cobblestone pattern of distribution of apical cells in A and apical and cytoplasmic immunogold particles in B. Altered distribution of binding of H185 antibody in non-Sjogren dry eye samples is demonstrated by immunofluorescence microscopy (C) and immunoelectron microscopy (D). Note loss of binding to apical cells in C, but enhanced bright spots of binding to goblet cells. Immunoelectron microscopy corroborates loss of binding to apical cells (D), but dense immunogold labeling to mucin packets (inset). Bars in A,C = 10 μm; Bars in B,D = 0.5 μm. (Reprinted from Gipson and Argüeso4 with permission of Int Rev Cytol.)

C. Glycosylation of Mucins: Alterations in Dry Eye

The importance of studying mucin glycosylation arises from the high numbers of clustered O-glycans on mucins, alterations of which may affect the physiology of mucins or mucous epithelia. Glycosyltransferases are the enzymes that transfer monosaccharide residues from their activated forms, the sugar nucleotides, to growing oligosaccharide chains on mucins. Glycosyltransferase efficiency is dictated by availability of donor sugars, which, in turn, is a function of tissue metabolic status and, therefore, removed from direct mucin genetic control. Mucin O-glycosylation is initiated post-translationally by the enzymatic addition of N-acetylgalactosamine (GalNAc) to serine and threonine residues (Figure 8A). The family of enzymes that catalyze this initial step, UDPGalNAc:polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), regulates the density and the position of Olinked sugar chains in the protein backbone.73 In the stratified epithelium of the normal human ocular surface, the distribution of members of the GalNAc-transferase family is celllayer and cell-type specific (Figure 8B).74 The highly ordered distribution of GalNAc-transferases observed in the conjunctiva of normal individuals is altered during the keratinization process, which may lead to altered glycosylation of mucins and severe ocular sur-

face dryness, such as occurs in OCP. There is increased GalNAc-transferase expression in the nonkeratinized conjunctival epithelium of OCP patients, suggesting that there is an initial attempt by the epithelium to maintain a wetsurfaced phenotype by upregulating or modifying mucintype O-glycosylation. A marked reduction in GalNActransferase antibody binding was observed in the late-stage, keratinized conjunctival epithelia of OCP patients, which may result in the aberrant synthesis of mucin O-glycans and, thus, in an alteration of the physicochemical properties of mucins. The biosynthesis of O-glycan structures following polypeptide GalNAc-transferase action is controlled by the repertoire of glycosyltransferases expressed by the cell, their level of activity, and their spatial organization over the Golgi apparatus.75 Elongation of the Tn antigen, as the mucin passes through the Golgi, generates up to eight different glycan core structures, the most common being core 1 and core 2 (Figure 8A).76 These core structures can then be extended by addition of polylactosamine and/or terminated by addition of one of a large repertoire of terminal carbohydrates (see example in Figure 8A). These extensions and terminations occur in a tissue- and cell-type-specific manner. Although there are no structural studies demonstrating the carbohydrate configuration of specific ocular mucins, it has been proposed that they contain mainly short

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oligosaccharide chains, such as Tn (GalNAcα1Ser/Thr) or sialyl Tn antigens (NeuAcα2-6GalNAcα1Ser/Thr) attached to the protein backbone.77 Other terminal carbohydraterelated epitopes, such as the blood group ABO and Lewis antigens (Figure 8A), have also been found within the goblet cell mucin packets and epithelial cells, indicating that mucin carbohydrate architecture may be more complex. Little is known regarding terminal sugars on ocular surface mucins, nor is it known whether each mucin type has unique carbohydrate characteristics. Several studies have demonstrated an altered expression of carbohydrates in dry eye syndrome.59,78 Versura et al, using a lectin-colloidal gold technique in combination with transmission electron microscopy, found alterations in the carbohydrate content of glycoconjugates of conjunctival goblet cells in dry eye patients as compared to normal subjects.78 In tears, Garcher et al have shown that a sialylated Lewis A carbohydrate epitope on glycoconjugates/mucins was significantly decreased in patients with dry eye syndrome.79 As stated above, immunohistochemical studies have also reported an alteration in the distribution of H185 mucin carbohydrate epitope carried on MUC16 in dry eye syndrome patients as compared to normal individuals (see below and Danjo et al, 199859). Although these data provide evidence of changes in mucin O-glycosylation in dry eye, the O-glycan composition of each mucin at the ocular surface is not known, nor is it known how the glycosylation of each individual mucin gene product is affected in dry eye. VI. DRY EYE THERAPEUTICS AND THEIR EFFECT ON MUCIN PRODUCTION/ DISTRIBUTION

The main therapy for dry eye is application of artificial tears. Recently, however, several drugs/agents have been reported to induce mucin expression or secretion by the human ocular surface epithelia. These may be good candidates as alternatives to artificial tears for treatment of dry eye, especially for the mucin-deficient type of dry eye. Recent data indicate that some of these drugs/agents regulate secreted mucins, while others regulate membrane-associated mucins. Since mucins are differentially regulated, perhaps application of reagents that affect both mucin types will be additive in their efficacy. A. Cyclosporine A

Recent studies suggest that dry eye syndrome is a disease associated with inflammation.60,80-83 For example, a significant increase in expression of the inflammatory markers, human lymphocyte antigen-DR (HLA-DR) and intercellular adhesion molecule-1 (ICAM-1) by conjunctival epithelial cells, was found in patients with dry eye syndrome.60,80 Cyclosporine A is an immunosuppressive agent commonly used systemically to treat inflammatory diseases, such as psoriasis or rheumatoid arthritis.84 Topical cyclosporine A has been used to treat corneal transplant rejection85 and Mooren’s ulcer.86 Recently, topical 142

Figure 8. A. Glycans commonly found attached to mucins. The biosynthesis of O-linked oligosaccharides or O-glycans is initiated by the enzymatic addition of N-acetylgalactosamine to serine or threonine residues to form the Tn antigen. Subsequent modification of the Tn antigen by addition of carbohydrate generates a variety of different configurations, such as sialyl-Tn antigen and the T antigen (Core 1). N-Acetylglucosamine can be linked to the T antigen to form the Core 2 structure, which can be further elongated by the sequential addition of lactosamine residues. A large number of terminal structures, such as the blood group ABH and Lewis antigens (e.g., sialyl-Lewis X), have been found on mucin oligosaccharide chains. Minor amounts of N-glycans containing the Trimannosyl core are also present on mucins.4 B. Diagram summarizing cell layer and goblet-cell-specific distribution of GalNAc transferases in human corneal and conjunctival epithelia. (Reprinted from Gipson and Argüeso4 with permission of Int Rev Cytol.)

cyclosporine A emulsion (RESTASIS™, Allergan Inc.; Irvine, CA) received approval from the FDA for patients with dry eye. Topical cyclosporine A treatment of dry eye patients has been reported to be clinically effective87,88 by reducing the presence of inflammatory markers in conjunctiva (HLA-DR and CD40).82 Kunert et al demonstrated that 6-month treatment with topical cyclosporine A (0.05%) not only reduced the number of cells expressing

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the lymphocyte activation markers CD11a and HLA-DR,89 but also increased the number of goblet cells in the conjunctival biopsy of patients with dry eye syndrome.69 Reducing inflammation of the ocular surface may lead to reconstitution of healthy conjunctival epithelial cells, resulting in an increase in the number of conjunctival goblet cells and a concomitant increase in secreted mucin MUC5AC in tears. B. P2Y2 Agonist

The P2Y2 receptor is a nucleotide receptor that is localized on diverse cell types, including epithelial, neuronal, glial, bone, and endothelial cells.90 At the ocular surface, the P2Y2 receptor gene is expressed by corneal and conjunctival epithelium of both rabbit and monkey, as determined by in situ hybridization.91 Jumblatt et al demonstrated that the exogenous P2Y2 nucleotide receptor agonists, adenosine 5’triphosphate (ATP) and uridine 5’triphosphate (UTP) stimulated mucin Figure 9. Diagram depicting an hypothesis that loss of hydrophilic secreted and membranesecretion in human and rabbit conjuncassociated mucins leads to loss of hydration and, in turn, to the formation of dry spots on tiva, as measured by lectin binding.92 apical cells of the ocular surface. Loss of the hydrophilic molecules leads to less retention The mechanism of this regulation is not of fluid, which, even with functional lacrimal glands, could lead to lower Schirmer values. (Reprinted from Argüeso and Gipson44 with permission of Exp Eye Res.) completely understood; however, the ATP- or UTP-associated induction of (S)-HETE may upregulate the expression of some of the memmucin-like secretions in the rat tracheal goblet cell line SPOC1 brane-associated mucins, but not the secreted mucins. The is reported to be regulated by Ca2++ and protein kinase Cexpression levels have not been measured; thus, it is not dependent pathways.93 Recently, a more stable P2Y2 recepknown if the reagent induces an increased release of MUC1 tor agonist, INS365 (Inspire Pharmaceuticals, Inc.; Durham, from cells or whether there is an increase in production or NC), was reported to stimulate tear secretion in a rat dry eye shedding of the mucin. It has been hypothesized that the model.94 This drug has been favorably evaluated in clinical trials (source: Inspire Pharmaceuticals, Inc) and is being dechange in MUC1 levels may be associated with the protein veloped for distribution by Allergan, Inc. It is not clear whether kinase C signal transduction pathway.95 the drug transiently affects goblet cell MUC5AC expression, D. Gefarnate or whether it affects shedding of the membrane-associated Gefarnate (3,7-dimethyl-2,6-octadienyl-5,9,13-trimethylmucins from the ocular surface. 4,8,12-tetradecatrienoate) is widely used for patients with C. 15-(S)-HETE gastric ulcer and gastritis. The mechanism of action of The eicosanoid, 15-(S)-hydroxy-5,8,11,13-eicosatetraegefarnate in the stomach is to normalize mucous secrenoic acid (HETE),95 is a metabolite of arachidonic acid by tion, which restores the defensive mucous barrier (as rethe lipoxygenase pathway. Previously, 15-(S)-HETE was viewed in Nakamura et al99). They reported that 1% gefarnate eye drops increased presence of mucin-like glyknown to stimulate secretion of mucus by airway epithecoproteins in media of cultured rat cornea, and, in an in lium.96,97 Jackson et al reported that topical application of 15-(S)-HETE to the rabbit ocular surface increased the thickvivo study, the drug reduced dessication in rabbit cornea100 allowed to dry by preventing eyelid closing with a specuness of the mucin layer on the surface of the corneal epithelum. Furthermore, treatment with the drug for 7 days inlium within 5 minutes, as measured by image analysis of eleccreased PAS-positive cell (goblet cell) density in rabbit tron micrographs.95 Furthermore, Jumblatt et al demonstrated that 15-(S)-HETE increased the amount of MUC1 protein, conjunctiva.99 Toshida et al demonstrated that treating a mild alkali injury to conjunctiva in a monkey model with but not MUCs 2, 4, 5AC, or 7 in human conjunctiva, as degefarnate for 4 weeks increased the number of goblet cells termined by dot-blot assay.64,98 These data suggest that 15THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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in the conjunctiva, as well as the amount of Muc5AC in tears.101 The rat corneal culture data suggest that gefarnate may induce shedding or expression of membrane-associated mucins, and the in vivo data suggest that goblet cell secretion and differentiation are also induced. E. Corticosteroids

Anti-inflammatory therapy using topical corticosteroids has recently been reported to be an efficacious therapy for patients with dry eye syndrome.102,103 Marsh et al reported the efficacy of topical administration of 1% nonpreserved methyprednisolone for patients with severe conjunctivitis sicca, demonstrating relief from irritation, a decrease in fluorescein staining, and resolution of filamentary keratitis.102,103 Avunduk et al compared treatment of KCS patients with the corticosteroid fluoromethalone (FML) to treatment with the nonsteroidal anti-inflammatory fluribiprofin.103 They found the corticosteroid to be more efficacious, producing the greatest decrease in symptom severity, fluorescein and rose bengal staining, increase in goblet cell numbers, and decrease in HLADR-positive cells. In terms of the effect of corticosteroids on mucin expression, membrane-associated mucin MUC1 mRNA is dramatically upregulated by a 24-hour incubation of immortalized human conjunctival epithelial (HCjE) cells with dexamethasone (1 μM), as determined by quantitative real-time PCR.104 Other mucin genes (4, 5AC, 16) were unaffected by dexamethasone. Perhaps upregulation of membrane-associated mucins lubricates the eye and leads to increased differentiation of goblet cells. F. Autologous Serum

Two clinical reports describe the efficacy of applying autologous serum for the treatment of dry eye due to Sjogren syndrome.105,106 Serum contains a number of growth factors, vitamin A, and anti-inflammatory factors that are needed for maintenance of a healthy ocular surface. We demonstrated that the expression of membraneassociated mucins MUCs 1, 4, and 16 were upregulated by serum in HCjE cells, both at the mRNA and protein level, as determined by quantitative real-time PCR and Western blot analysis, respectively.107 We also found that the pattern of regulation of each membrane-associated mucin was different from the others, suggesting that the three mucins are independently regulated. These data suggest that the efficacy of autologous serum application for dry eye may be related to the upregulation of expression of these three membrane-associated mucins in human ocular surface epithelia. Study of the alteration of the expression of secreted mucin has not been performed. G. Vitamin A

It is well known that the ocular surface has an absolute requirement for vitamin A. Lack of sufficient vitamin A causes abnormal differentiation of the ocular surface, resulting in keratinization of both conjunctival and corneal epithelial cells.108 Depletion of vitamin A in the diet of rats caused loss of expression of rMuc5AC and rMuc4 144

in ocular surface epithelia, whereas rMuc1 levels were unaffected.72 Topical vitamin A has been reported to be effective as a treatment for severe squamous metaplasia,109,110 but not for keratoconjunctivitis sicca.110,111 We reported that treatment with 100 nM retinoic acid, the biologically active form of vitamin A, in the absence of serum upregulated the expression of both MUC4 and MUC16 mRNA and protein in HCjE cells, whereas MUC1 was unaffected.107 These data may explain the efficacy of retinoic acid (vitamin A) for severe mucin-deficient squamous metaplasia. VIII. SUMMARY: HYPOTHESIS REGARDING MUCIN ALTERATIONS IN DRY EYE

With the characterization of expression of mucins by the ocular surface epithelia, studies of their alteration in ocular disease have begun. These studies have required development of new techniques to assay changes in expression level of mucin genes, content of mucins in tears, and measurements of their post-translational modification. To date, decreases in levels of MUC5AC mRNA and tear protein have been demonstrated in patients with Sjogren syndrome. Glycosylation also seems to be altered on membrane-associated mucins; distribution of the H185 carbohydrate epitope on MUC16 is altered in non-Sjogren dry eye. Based on these early findings, as well as on new information that goblet cell mucin moves over an epithelial surface glycocalyx that is composed of membrane-associated mucins, which are especially prevalent in microplicae, we have developed an hypothesis regarding drying at the ocular surface. Decreased production of MUC5AC, as well as changes in membrane-associated mucins (either downregulation, altered glycosylation, or increased shedding) leads to loss of microplicae and less tear adherence to epithelial cells on localized regions of the ocular surface (Figure 9). These “dry spots” stain with rose bengal and lead to shorter tear breakup times. The loss of the highly hydrophilic mucin population implies that, even if lacrimal fluid is available, it will not be retained at the ocular surface. REFERENCES 1. Gendler SJ, Spicer AP. Epithelial mucin genes. Annu Rev Physiol 1995;57:607-34 2. Moniaux N, Escande F, Porchet N, et al. Structural organization and classification of the human mucin genes. Front Biosci 2001;6:D1192-206 3. Fleiszig SM, Zaidi TS, Ramphal R, Pier GB. Modulation of Pseudomonas aeruginosa adherence to the corneal surface by mucus. Infect Immun 1994;62:1799-1804 4. Gipson IK, Argueso P. The role of mucins in the function of the corneal and conjunctival epithelia. Int Rev Cytol 231:1-49 5. Sellers LA, Allen A, Morris ER, Ross-Murphy SB. The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components. Biochim Biophys Acta 1991;1115:174-179 6. Desseyn JL, Buisine MP, Porchet N, et al. Evolutionary history

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